Oxidative stresses‐mediated apoptotic effects of ginsenoside Rb1 on pre‐ and post‐implantation mouse embryos in vitro and in vivo

Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side‐effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo . Blastocysts treated with 25–100 μg mL^?1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25–100 μg mL^?1 ginsenoside Rb1 was associated with increased resorption of post‐implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mg kg^?1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses‐triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre‐ and post‐implantation development of mouse embryos, both in vitro and in vivo . © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990–2003, 2017.     

Effect of genistein on mouse blastocyst development in vitro

AIM: To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro. METHODS: Mouse blastocysts were incubated in medium with or without genistein (25 or 50 micromol/L) or daidzein (50 micromol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis. RESULTS: TUNEL staining and Annexin-V/PI staining showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment decreased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis. CONCLUSIONS: Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein on mouse blastocysts.     

Berberine impairs embryonic development in vitro and in vivo through oxidative stress‐mediated apoptotic processes

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H₂O₂‐induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine—triggering or preventing apoptosis—has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5‐10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine‐induced injury of mouse blastocysts appeared to be attributable to oxidative stress‐triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Cytotoxic effect of CdSe quantum dots on mouse embryonic development

AIM: The aim of this study was to examine the cytotoxic effect of quantum dots (QD), a novel luminescent material, on early post-implantation embryonic development. METHODS: Mouse blastocysts were incubated in medium with or without CdSe-core QD (250 or 500 nmol/L) for 24 h. Cell apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay and Annexin V/propidium iodide staining, and proliferation was investigated by dual differential staining. Pre-implantation and post-implantation development was assessed by in vitro and in vivo analyses, respectively. RESULTS: The apoptotic staining analysis showed that CdSe-core QD induced apoptosis in mouse blastocysts in a dose-dependent manner. Pretreatment of blastocysts with CdSe-core QD inhibited cell proliferation, primarily in the inner cell mass. CdSe-core QD also inhibited post-implantation embryonic development; fewer CdSe-core QD-pretreated blastocysts reached the later stages of development compared to the controls. The pre-implantation development of morulas into blastocysts was also inhibited by CdSe-core QD. Furthermore, CdSe-core QD at 500 nmol/L were associated with resorption of post-implantation blastocysts and a decrease in fetal weight. The cytotoxicity of CdSe QD in embryonic development was significantly reduced by the addition of a ZnS coating. CONCLUSION: Our results show that CdSe-core QD induce apoptosis in mouse blastocysts, inhibit cell proliferation, retard early post-implantation blastocyst development, and increase early-stage blastocyst death in vitro and in vivo.     

Prevention of ochratoxin A‐induced oxidative stress‐mediated apoptotic processes and impairment of embryonic development in mouse blastocysts by liquiritigenin

Ochratoxin A (OTA), a mycotoxin constituent of a range of food commodities, including coffee, wine, beer, grains, and spices, exerts toxicological and pathological effects in vivo, such as nephrotoxicity, hepatotoxicity, and immunotoxicity. In a previous report, we highlighted the potential of OTA to induce apoptosis via reactive oxygen species (ROS) generation in mouse blastocysts that led to impaired preimplantation and postimplantation embryo development in vitro and in vivo. Here, we have shown that liquiritigenin (LQ), a type of flavonoid isolated from Glycyrrhiza radix, effectively protects against OTA‐mediated apoptosis and inhibition of cell proliferation in mouse blastocysts. Preincubation of blastocysts with LQ clearly prevented OTA‐triggered impairment of preimplantation and postimplantation embryonic development and fetal weight loss, both in vitro and in vivo. Detailed investigation of regulatory mechanisms revealed that OTA mediated apoptosis and embryotoxicity through ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase‐9 and caspase‐3, which were effectively prevented by LQ. The embryotoxic effects of OTA were further validated in an animal model in vivo. Intravenous injection of dams with OTA (3 mg/kg/day) led to apoptosis of blastocysts, impairment of embryonic development from zygote to blastocyst stage and decrease in day 18 fetal weight. Notably, preinjection of dams with LQ (5 mg/kg/day) effectively prevented OTA‐induced apoptosis and toxic effects on embryo development. Our collective results clearly demonstrate that OTA exposure via injection has the potential to damage preimplantation and postimplantation embryonic development against which LQ has a protective effect.     

Embryonic toxicity of sanguinarine through apoptotic processes in mouse blastocysts

In this study, we examined the cytotoxic effects of sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation via embryo transfer. Blastocysts treated with 0.5-2 μM sanguinarine exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with sanguinarine were lower than that of their control counterparts. Moreover, in vitro treatment with 0.5-2 μM sanguinarine was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that sanguinarine induces apoptosis and retards early post-implantation development in vitro and in vivo. In addition, sanguinarine induces apoptotic injury effects on mouse blastocysts through intrinsic and extrinsic apoptotic signaling processes to impair sequent embryonic development. However, the extent to which sanguinarine exerts teratogenic effects on early human development is not known at present, and further studies are required to establish effective protection strategies against its cytotoxic effects.     

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune‐stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside‐Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25–100 μM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1‐treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 μM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst‐stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress‐mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl‐2 ratio, and activates caspase‐9 and caspase‐3, but not caspase‐8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.     

Effect of citrinin on mouse embryonic development in vitro and in vivo

Citrinin (CTN), a mycotoxin that is often found as a natural contaminant in foodstuffs and animal feeds, has been demonstrated to have cytotoxic and genotoxic effects on various mammalian cells. In this study, we examined the cytotoxic effects of CTN on mouse blastocysts and subsequent early development in vitro and in vivo. Blastocysts treated with 15 or 30 microM CTN showed significant increases in apoptosis and significant decreases in total cell number. In addition, CTN-pretreated blastocysts showed a significantly lower implantation success rate. Treatment with 30 microM CTN was associated with increased resorption of postimplantation embryos and decreased fetal weight. Our results collectively indicate that CTN-induced apoptosis in the mouse blastocyst reduced cell number and retarded early postimplantation development. The extent to which CTN may have teratogenic potential in early human development is not known.     

Induction of cytotoxicity and apoptosis in mouse blastocysts by silver nanoparticles

Silver nanoparticles (nanoAg) are antibacterial materials widely used in various products and medical supplies. In this report, we examined the cytotoxic effects of nanoAg on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 50 μM nanoAg exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Importantly, the implantation success rate of blastocysts pretreated with nanoAg was lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM nanoAg was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that in vitro exposure to nanoAg induces apoptosis and retards early post-implantation development after transfer to host mice. However, nanoAg-stimulated embryonic cytotoxicity appeared lower than that induced by the Ag⁺ ion. The results collectively show that nanoAg has the potential to induce embryo cytotoxicity. Further studies are required to establish effective protection strategies against the cytotoxic effects of these nanoparticles.     

Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts

Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca(2+) and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells.     

Effects of 3H-thymidine on preimplantation mouse embryos in vitro

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.     

Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes

Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N‐acetylcysteine effectively prevented AgNP‐triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP‐induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac‐DEVD‐cho, a caspase‐3‐specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase‐dependent apoptotic signaling cascades in AgNP‐mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP‐pretreated oocytes via intracellular ROS generation, which is further mediated through p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms.     

Injury effects of ginkgolide B on maturation of mouse oocytes,fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1–6 μM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3–6 μM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.     

Dosage‐related beneficial and deleterious effects of ginsenoside Rb1 on mouse oocyte maturation and fertilization and fetal development

Ginsenoside Rb1 (GRb1), the major saponin component of ginseng root, has a wide range of therapeutic applications for various diseases. Previously, our group showed that GRb1 triggers ROS‐mediated apoptotic cascades in mouse blastocysts, leading to decreased cell viability and impairment of pre‐ and postimplantation embryonic development, both in vitro and in vivo. In this study, we further found that GRb1 exerted dose‐dependent effects on oocyte maturation and sequent development in vitro. Oocytes preincubated with 25 μg/mL GRB1 displayed significantly enhanced maturation and in vitro fertilization (IVF) rates, along with progression of subsequent embryonic development. In contrast, treatment with 50 and 100 μg/mL GRB1 led to impairment of mouse oocyte maturation, decreased IVF rates, and injurious effects on subsequent embryonic development. In vivo, intravenous injection of 1 mg/kg body weight GRb1 significantly promoted mouse oocyte maturation, IVF, and early‐stage embryo development after fertilization while administration of 5 mg/kg body weight GRb1 led to a marked decrease in oocyte maturation and IVF rates concomitant with impairment of early embryonic development in our animal model. In terms of the mechanisms underlying the regulatory effects of GRb1 demonstrated increased intracellular reactive oxygen species (ROS) production and apoptosis in the 100 μg/mL GRb1 treatment group. However, we observed a significant decrease in total intracellular ROS content and inhibition of apoptosis events in the 25 μg/mL GRb1 treatment group, signifying that the intracellular ROS content serves as a key upstream regulator of GRb1 that influences its dose‐dependent beneficial or deleterious effects on oocyte maturation and sequent embryonic development. For further clarification of the mechanisms underlying GRb1‐triggered injurious effects, oocytes were pretreated with Ac‐DEVD‐CHO, a caspase‐3‐specific inhibitor, which effectively blocked injury to oocyte maturation, fertilization, and sequent development. In sum, study findings highlight the potential involvement of p53‐, p21‐, and caspase‐3‐dependent regulatory signaling cascades in GRb1‐mediated apoptotic processes.     

Effect of nickel chloride and cadmium acetate on the development of preimplantation mouse embryos in vitro

Preimplantation mouse embryos were used to investigate the toxic effect of nickel chloride and cadmium acetate on early embryo development in vitro.Embryos at the 2- and 4–8 cell stage were cultured in approximately 0.05 ml of mouse embryo culture medium (No. 16), overlaid with paraffin oil and incubated in a humidified atmosphere of 5% CO₂ in air for 48 h. NiCl₂ · 6H₂O was added to the culture medium at concentrations of 10–1000 μM, Cd(CH₃COO)₂ · 2H₂O at concentrations of 10–50 μM. Morphological criteria were used to check embryonic development.Ten micromolars of nickel chloride affected adversely the development of Day 2 embryos (2-cell stage), whereas 300 μM was needed to affect Day 3 embryos (8-cell stage). Toxic effect of cadmium acetate on Day 2 embryos was observed at a concentration of 10 μM.     

Effects of ochratoxin a on mouse oocyte maturation and fertilization,and apoptosis during fetal development

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1–10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked these OTA‐triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase‐dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA‐pretreated oocytes, indicating that such cells undergo apoptosis via p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724–735, 2016.     

维生素A酸对小鼠囊胚细胞凋亡的影响

目的:探讨全反式维生素A酸(ATRA)对体外培养小鼠囊胚中内细胞群(ICM)和滋养层(TE)细胞凋亡的影响。方法:获取妊娠3.5d小鼠囊胚,分别培养在含0μmol/l、1μmol/l和10μmol/l的ATRA的M199培养基中24h,用带有荧光(FITC)标记的原位末端标记(TUNEL)检测法,观察ATRA对小鼠囊胚细胞凋亡的效应。结果:ATRA可诱导囊胚细胞凋亡,并增强小鼠囊胚Fas蛋白表达。结论:维生素A酸对小鼠胚胎的发育具有细胞毒性作用。     

In vitro exposure of male and female mice gametes to cadmium chloride during the fertilization process,and its effects on pregnancy outcome

The effect of cadmium chloride (Cd) on gamete fusion in vitro was evaluated, with further observations of the embryonic development and assessment of the pregnancy outcome of the in vitro fertilized mice. Oocytes were recovered from superovulated B₆C₃F₁ female mice and inseminated in vitro with spermatozoa from B₆C₃F₁ males. Of 1210 control oocytes, 53.2% cleaved into two-cell stage embryos. Of these, 46.6% developed into blastocyst stage embryos which were then surgically transferred to pseudopregnant female CD-1 mice. Of a total of 63 implanted embryos, 8 (12.7%) developed in utero to live fetuses. Teratological examinations of these “test-tube” mice revealed no signs of abnormalities caused by in vitro culture. Male and female gametes were exposed to 0.4, 0.8, or 1.6 μm of Cd and a decrease in sperm motility was noted in the 1.6 μm group. Nevertheless, even in the highest concentration used, 56.4% of the ova cleaved into the two-cell stage, thus indicating no effect of Cd on initial gamete interaction. Gametes that had been treated with 0.4 and 0.8 μm Cd developed to blastocysts at rates comparable to that of the controls. In the 1.6 μm group, however, only one (3.2%) of the two-cell embryos developed to the blastocyst stage. Blastocysts from 0.4 μm Cd-treated gametes were then transferred to surrogate dams. Statistically significant blastocyst losses were recorded during the implantation period, whereas the pregnancy rate and the numbers of resorbed and live fetuses, were comparable to those of the controls. The offspring exhibited no malformations, and their body weights remained within the control values.